肿瘤坏死因子-α通过激活A549细胞Wnt/β-catenin通路降低肺泡液体清除功能  

Tumor Necrosis Factor-α Reduces Alveolar Fluid Clearance through Activation of the Wnt/β-catenin Pathway in A549 Cells

作　　者：李美 马希刚[2] LI Mei;MA Xigang(School of Clinical Medicine Sciences,Ningxia Medical University,Yinchuan 750004,China;Department of Critical Care Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏医科大学总医院重症医学科,银川750004

出　　处：《宁夏医科大学学报》2023年第2期124-130,137,共8页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金重点项目(2022AAC02064);宁夏回族自治区重点研发项目(2020BEG03012)。

摘　　要：目的探讨炎症环境下Wnt/β-catenin通路对肺泡液体清除功能的影响。方法用不同浓度(10、40、80、160 ng·mL^(-1))肿瘤坏死因子-α(TNF-α)处理人肺泡上皮Ⅱ型细胞(A549)24 h,采用CCK-8法检测细胞存活率,酶联免疫吸附实验(ELISA)检测炎性因子白介素(IL)-1β、IL-18、IL-6表达水平,Western blot检测钠水转运蛋白上皮细胞钠通道β亚型(βENaC)、NA^(+)、K^(+)-ATP酶(NKA)和水通道蛋白(AQP)4的表达,综合实验结果选取TNF-α最佳干预浓度;然后A549分为control组、TNF-α40 ng·mL^(-1)组、Wnt通路抑制组(TNF-α+XAV939组),分别检测细胞存活率、凋亡率、炎性因子表达水平及β-连环蛋白(β-catenin)、细胞周期蛋白D1(Cyclin D1)、腺瘤息肉病基因蛋白(APC)、βENaC、NKA和AQP4的表达水平。结果自TNF-α的40 ng·mL^(-1)起,A549细胞存活率逐渐降低,炎性因子表达水平逐渐升高,钠水转运蛋白βENaC、NKA表达逐渐降低,AQP4表达逐渐升高(P<0.05),选出最佳干预浓度为TNF-α40 ng·mL^(-1);与control组相比,TNF-α40 ng·mL^(-1)组A549细胞存活率降低,凋亡率、炎性因子表达水平升高,βENaC、NKA、APC蛋白表达降低,AQP4、β-catenin、Cyclin D1蛋白表达升高(P均<0.05)。应用XAV939后,与TNF-α40 ng·mL^(-1)组相比,TNF-α+XAV939组A549细胞存活率升高,凋亡率、炎性因子表达水平降低,βENaC、NKA、APC蛋白表达升高,AQP4、β-catenin、Cyclin D1蛋白表达降低(P均<0.05)。结论TNF-α可通过激活A549细胞Wnt/β-catenin通路,使钠水转运蛋白βENaC、NKA表达减少,AQP4表达增加,降低肺泡液体清除功能。Objective To investigate the effect of Wnt/β-catenin pathway on alveolar fluid clearance function in inflammatory environment.Methods Human alveolar epithelial typeⅡcells(A549)were treated with different concentrations of tumor necrosis factor-α(TNF-α:10,40,80,160 ng·mL^(-1))for 24 h.Cell survival rates were detected by CCK-8,inflammatory factors IL-1β,IL-18 and IL-6 levels were detected by ELISA,and sodium-water transport proteinsβENaC,NA^(+)NKA and AQP4 were detected by Western blot.The optimal intervention concentration of TNF-αwas selected by comprehensive experimental results.The cells were divided into control group,TNF-α40 ng·mL^(-1) group and Wnt pathway inhibition group(TNF-α+XAV939 group).And cell survival rate,apoptosis rate,inflammatory factor level and the expression levels ofβ-catenin,Cyclin D1,APC,βENaC,NKA and AQP4 were detected respectively.Results From 40 ng·mL^(-1) of TNF-α,A549 survival rate gradually decreased,inflammatory factor expression level gradually increased,sodium water transport proteinβENaC and NKA expression gradually decreased,AQP4 expression gradually increased(P<0.05),and the optimal intervention concentration was selected as TNF-α40 ng·mL^(-1);compared with the control group,the TNF-α40 ng·mL^(-1),A549 survival rate decreased,apoptosis rate and inflammatory factor expression level increased,βENaC,NKA and APC proteins expression decreased,and AQP4,β-catenin and Cyclin D1 proteins expression increased(P all<0.05).After applying XAV939,compared with the TNF-α40 ng·mL^(-1) group,the TNF-α+XAV939 group had increased A549 survival rate,decreased apoptosis rate and inflammatory factor expression level,increasedβENaC,NKA,APC proteins expression,and decreased AQP4,β-catenin,Cyclin D1 proteins expression(P all<0.05).Conclusion TNF-αdecreases alveolar fluid clearance function in A549 by activating the Wnt/β-catenin pathway,resulting in decreased expression of sodium-water transporter proteinsβENaC and NKA and increased expression of AQP4.

关 键 词：急性肺损伤 急性呼吸窘迫综合征 WNT/Β-CATENIN通路 水通道蛋白 钠通道蛋白 钠钾泵 

分 类 号：R563[医药卫生—呼吸系统] R34[医药卫生—内科学]